We have discovered that an Escherichia coli protein named NleB, which is highly conserved and expressed by the attaching/effacing (A/E) bacterial pathogens of significance to human health, is a potent inhibitor of host interferon (IFN) production. IFN was originally discovered as a factor secreted by virally infected cells able to mediate protection of other cells from subsequent viral infection. It has become convincingly clear that type I IFNs are important not only to anti-viral defense but also to host defense against bacterial infection. Diarrheal disease caused by enteric bacteria is a major endemic health threat and an increasingly frequent source of foodborne illness. Among these enteric pathogens, enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC) are frequent causes of infantile diarhea and hemorhagic colitis, respectively. EHEC is especially significant because it is a leading cause of pediatric renal failure. While it is known that bacterial and viral pathogens have evolved strategies to interfere with mammalian type I IFN signaling, the extent to which bacterial effector proteins translocated by type III secretion systems (T3SS), an important virulence strategy of Gram-negative pathogens, are able to inhibit type I IFN production, has not been observed previously. These studies will elucidate, for the first time, the mechanism by which a T3SS effector inhibits IFN-? production in infected cells. The specific aims are to: 1) Characterize the host protein(s) to which NleB binds that results in decreased IFN-? production in cells infected by attaching/effacing pathogens. 2) Determine the impact of NleB and infection by attaching/effacing pathogens on the differential activation vs. degradation-associated ubiquitination of TBK1, a host protein kinase essential to activation of type I IFN responses. The proposed experiments will have a significant positive impact on human health because they will allow us to elucidate a novel mechanism by which enteric pathogens promote their survival by suppressing the host innate immune response. First, determining the host target of and molecular mechanism by which NleB inhibits IFN-? production will provide an important advance in our understanding of T3SS effector targets and mechanisms during infection. Notably, interplay between T3SS effectors and the type I interferon pathway has not been previously reported. Second, elucidating NleB function may suggest novel therapeutic strategies to combat bacterial diarrheal diseases. Additionally, our studies may improve the understanding of and eventually improve the treatment for viral-bacterial co-infections, as NleB-mediated suppression of type I IFN has the potential to exacerbate diarrheal illness mediated by viruses with gastrointestinal tropism. PUBLIC HEALTH RELEVANCE: Escherichia coli causes potentially fatal diarrheal disease and is transmitted to humans via contaminated food and water. A group of antimicrobial proteins known as interferons is important to controlling bacterial infection. We have discovered that E. coli injects into infected intestinal cells a protein named NleB that is a potent inhibitor of the interferon response. The focus of this proposal is to determine the mechanism of action used by NleB to inhibit host interferon production. Data obtained from these studies may improve our ability to prevent and treat bacterial infections.